The Studies On The Molecular Mechanisms Of IL-2 Muteins

Interleukin-2(IL-2)was originally discovered as a T cell growth factor(TCGF)in 1976 and was first cloned in 1983.As the first cytokine effectual in cancer immunotherapy,IL-2 signals can stimulate different lymphocyte subsets,including T,B and natural killer(NK)cells,during their proliferation,differentiation and immune responses.Remarkably,mass of evidence demonstrates that IL-2 is essential for the immune homeostasis and balance of Treg and Teff cells in the immune system,especially for cell number maintenance and functional activity of Treg cells.Thus,the administration of IL-2 is considered as an effective method to boost Treg cell numbers and function to treat autoimmune diseases.However,the short half-life,IL-2-induced toxicity and off-target effects on different cell populations limit the therapeutic application of IL-2 for cancer or autoimmune disorders.In recent years,other approaches like IL-2/m Ab complexes,IL-2 muteins and low-dose-IL-2 emerged and revived IL-2 therapeutic strategies.Levin and his colleagues engineered H9(known as “superkine”)which increased the binding affinity to IL-2Rβ and improved anti-tumor responses.Based on superkine H9,Mitra and his colleagues engineered new H9 muteins such H9-T,H9-RE,H9-RET and H9-RETR as receptor signaling clamps which acted as partial agonists or complete antagonist of wild-type(WT)IL-2 to control IL-2/receptors signaling.But the effects of muteins on three signaling pathways of IL-2 were not fully investigated.To further analyze the effects of muteins on IL-2 signaling pathways and explore the molecular mechanism of muteins by analyzing dynamic changes of phosphorylation at cellular level,WT IL-2 and muteins H9-RE,H9-RET and H9-RETR with two Flag tags at C-terminus(noted as DF-WT IL-2,DF-H9,DFH9-RE,DF-H9-RET and DF-H9-RETR)were constructed to explore their biological function of muteins based in cultured CTLL-2 cells in this study.The effects of muteins on cell viability were detected by CTG.Moreover,the antagonistic functions of DFH9-RET and DF-H9-RETR on WT IL-2 were also analyzed by competition studies.Results in competition experiments demonstrated that DF-H9-RET also acted as an IL-2 antagonist like DF-H9-RETR,which had implications for therapy of autoimmune diseases.In addition,we detected mutein-induced phosphorylation of MAK kinase and P3IK-AKT signaling pathway which were not studied in Mitra’s report.The results revealed that phosphorylation patterns in MAK kinase,PI3K-AKT and JAK-STAT5 signaling pathways induced by different muteins were distinct.DF-H9 had the same capacity to phosphorylated signal molecules as WT IL-2 do,DF-H9-RE was a weakeractivator and DF-H9-RET and DF-H9-RETR lost their capacities to transduce signals.Therefore,IL-2 muteins not only altered the cell proliferation,but also the signaling.Dynamic changes of the signaling by time course studies showed the variation trend of phosphorylation and dephosphorylation over time in each signaling pathway.It further explained the mechanisms underlying the functions of H9 and H9-RETR in vivo,which suggested that H9 led to improved anti-tumor responses because of its bias to Teff cells,while DF-H9-RET and DF-H9-RETR suppressed targeting cells activities.The DF-H9-RE could be used as the substitution of WT IL-2 with moderate functions.DF-H9-RET and DF-H9-RETR could inhibit the Teff,and be potential therapeutic agents in autoimmune disease treatments.In summary,the molecular mechanism studies of cell proliferation,signaling pathway and dynamic analysis of IL-2 muteins provided a solid foundation for designing novel IL-2 mutants and analyzing their mechanism.